Dynamics of SAS-I mediated H4 K16 acetylation during DNA replication in yeast

Abstract

The acetylation of H4 lysine 16 (H4 K16Ac) in Saccharomyces cerevisiae counteracts the binding of the heterochromatin complex SIR to chromatin and inhibits gene silencing. Contrary to other histone acetylation marks, the H4 K16Ac level is high on genes with low transcription, whereas highly transcribed genes show low H4 K16Ac. Approximately 60% of cellular H4 K16Ac in S. cerevisiae is provided by the SAS-I complex, which consists of the MYST-family acetyltransferase Sas2, Sas4 and Sas5. The absence of SAS-I causes inappropriate spreading of the SIR complex and gene silencing in subtelomeric regions. Here, we investigated the genome-wide dynamics of SAS-I dependent H4 K16Ac during DNA replication. Replication is highly disruptive to chromatin and histone marks, since histones are removed to allow progression of the replication fork, and chromatin is reformed with old and new histones after fork passage. We found that H4 K16Ac appears in chromatin immediately upon replication. Importantly, this increase depends on the presence of functional SAS-I complex. Moreover, the appearance of H4 K16Ac is delayed in genes that are strongly transcribed. This indicates that transcription counteracts SAS-I-mediated H4 K16 acetylation, thus "sculpting" histone modification marks at the time of replication. We furthermore investigated which acetyltransferase acts redundantly with SAS-I to acetylate H4 K16Ac. esa1Δ sds3Δ cells, which were also sas2Δ sir3Δ in order to maintain viability, contained no detectable H4 K16Ac, showing that Esa1 and Sas2 are redundant for cellular H4 K16 acetylation. Furthermore, esa1Δ sds3Δ sas2Δ sir3Δ showed a more pronounced growth defect compared to the already defective esa1Δ sds3Δ sir3Δ. This indicates that SAS-I has cellular functions beyond preventing the spreading of heterochromatin.

Fig 1. Establishment of an auxin-regulated version of the SAS-I component SAS4.

(A) SAS4-AID (SAS4-AID) downregulation by addition of auxin (OFF) caused a reduction of bulk H4 K16Ac. Top, Detection of myc-tagged Sas4-miniAID by Western blotting with an α-myc antibody; middle, α-H4 K16Ac; bottom, GAPDH (loading control). (B) Sas4-AID provided Sas4 function in HML silencing. Semi-quantitative mating of the indicated MATa strains in the presence or absence of auxin is shown. (C) Reduction of H4 K16Ac levels by downregulation of Sas4-AID at subtelomeric regions on the right arm of telomere VI (TEL-VIR) and selected genes, as measured by ChIP. Means of two independent biological replicates of H4 K16Ac relative to H4 are shown.

Fig 2. Replication-coupled acetylation of H4 K16 depends on the SAS-I histone acetyltransferase complex.

(A) Experimental set-up to measure the dynamics of H4 K16Ac during S-phase. Cells were arrested in S-phase with HU for 1.5 hours; Sas4-AID was subsequently depleted by the addition of auxin (+IAA) for 30 minutes in the samples SR+IAA and NR+IAA (the solvent ethanol is added to the control experiment, SR). Cells of the SR+IAA and SR samples were then collected by centrifugation and resuspended in medium lacking HU. Samples were collected in 10 minute intervals and processed for FACS (B), Western blotting (C) and ChIP-seq (D). (C) Monitoring the depletion of Sas4-miniAID and H4 K16Ac in the synchronization/ release experiments. Blots were performed as in Fig 1A. Sync, synchronization. (D) Replication-associated dynamics of H4 K16Ac. The abundance of H4 K16Ac along chromosome XI is shown. Each plot gives the signal along the chromosome (horizontal) at the different times (vertical) relative to the average of the entire time-course. SR, Sas4-AID is on; SR+IAA, Sas4-AID is off; bottom, NR+AID, Sas4-AID is off, but cells were maintained in S-phase (“no release”, see A). Bottom, replication profile of chromosome XI as previously determined [26]. Replication time is plotted as a function of the coordinate.

Fig 3. Genome-wide dynamics of H4 K16Ac during replication.

H4 K16Ac levels during replication were measured in the presence (A, SR) and absence (B, SR+IAA) of Sas4. Data is represented as in Fig 2D.

Fig 4. Dynamics of H4 K16Ac on origins of replication during replication.

H4 K16Ac increased later on late-replicating origins. (A) Profiles of H4 K16Ac 2 kB upstream and downstream of ARS sequences. Origins were grouped according to their time of replication (from left to right: the earliest 25% of ARS (“early”); second earliest origins (25–50%, “early—mid”); later origins (50–75%, “mid—late”); latest origins (“late”). (B) H4 K16Ac was averaged over the cluster of ARS (ARS +/- 2 kB, but excluding 500 bp surrounding the ARS) and across the time courses.

Fig 5. Dynamics of H4 K16Ac on genes of varying expression level.

(A) The average H4 K16Ac in the expression clusters is plotted relative to the distance to the transcription start site (TSS). Genes were grouped into clusters based on gene expression in HU-arrested cells (0–25%, lowest expression; 75–100%, highest expression). (B) Genes with high expression showed a delay in replication-mediated increase of H4 K16Ac. H4 K16Ac was averaged across the genes of the cluster, but excluding the first 350 bp. Signals were adjusted to the same dynamic range. (C, D) Replication-associated increase of H4 K16Ac is more prominent in long compared to short genes. (C) Profiles were generated as in A, except that genes were selected by length (500–1000 bp; 1000–1500 bp; 1500–2000 bp; > 2000 bp). (D) Increase of H4 K16Ac during replication occurs earlier on longer compared to shorter genes. Representation as in B.

Fig 6. Yeast lacking both SAS-I and Esa1 show no detectable H4 K16Ac.

(A) Absence of Sas2 caused a further decrease of viability in esa1Δ sds3Δ cells. Strains with the indicated genotypes were serially diluted and spotted on complete medium. Plates were incubated at the indicated temperatures for 2–3 days. Strains were sir3Δ in order to circumvent the lethality of sas2Δ with sds3Δ. H4 K16Ac was undetectable in esa1Δ sas2Δ cells by Western blotting (B, as in Fig 1A) and ChIP (C). (C) shows H4 K16Ac relative to H4 (mean ± SD of three independent biological replicates).